1. Field of the Invention
The invention relates to antibodies that bind to a fragment of the Response Gene to Complement-32 (RGC-32), as well as methods of using these antibodies.
2. Background of the Invention
Progression through each phase of the cell cycle is controlled by specific cyclin dependent kinases (CDK) and their interactions with cyclins and CDK inhibitors (CKI). The expression of each cyclin fluctuates throughout the cell cycle, and CKI are down-regulated in response to mitogenic stimulation (Sherr C. J. Science, 274:1672-1677 (1996), which is incorporated by reference). CDK are a family of serine/threonine protein kinases that are regulated by multiple mechanisms leading to their activation at specific points of the cell cycle. Mitosis is regulated by CDC2 when in complex with cyclin B (Nigg, E. A., Nat. Rev. Mol. Cell. Biol., 2, 21-32 (2001), incorporated by reference). Deregulation of the cell cycle is well documented in cancer (See Sherr, C. J., Cancer Res., 60, 3689-3695 (2000), which is incorporated by reference), and compounds with CDK inhibitory activity have recently entered clinical trials (See Vermeulen, K., et al., Cell Prolif. 36: 131-149 (2003), which is incorporated by reference). The expression of CDC2, CDK2 and CDK4 proteins is higher in colon cancer cells than in normal mucosa (See Kim, J. H. et al., Cancer, 85: 546-553 (1999) and Salh, B., et al., Anticancer Res., 19:741-748 (1999), which are incorporated by reference.) Higher levels of cyclins D1, D3, A and E were also found in primary colorectal carcinomas than in the adjacent normal areas (See Arber, N., et al., Gastroenterology, 110: 669-674 (1996), Handa, K., et al., Int J Cancer, 84: 225-233 (1999) and Sutter, T., et al., J Med, 28: 285-309 (1997), all of which are incorporated by reference). CDC2 kinase activity is also documented to be increased in colon cancer tissue, but not in normal tissue. CDC2 is mostly present in colon cancer cells positive for phosphorylated Rb protein (Yamamoto, H., et al., Br. J. Cancer., 71: 1231-1236 (1995), which is incorporated by reference), and its overexpression is higher in focal carcinomas (Yamamoto, H., et al., Int J Oncol, 13: 233-239 (1998), which is incorporated by reference). The cyclin dependent pathway is, however, complicated and other factors are necessary for proper function and progression through the cell cycle.
One of these other factor, the Response Gene to Complement (RGC)-32 was first cloned in the rat by differential display (See Badea, T. et al., J. Biol. Chem., 273:266977-26981 (1998), which is incorporated by reference), and subsequently from human brain library (See Badea, T. et al., J. Biol. Chem. 277:502-508 (2005), which is incorporated by reference). Overexpression of RGC-32 is associated with an increase in DNA synthesis, thus leading to the hypothesis that RGC-32 is involved in activation of the cell cycle Badea, T. et al., J. Biol. Chem., 273:266977-26981 (1998). Experimental evidence indicates that RGC-32 has an important role in cell cycle activation through regulation of CDC2 kinase (Badea, T. et al., J. Biol. Chem. 277:502-508 (2005)). Overexpression of RGC-32 in human aortic smooth muscle cells (SMC) increased BrdU incorporation and the number of cells progressing into G2/M phase. RGC-32 appears to complex with CDC2/cyclin B1 and increase the kinase activity of CDC2. This kinase-enhancing activity requires CDC2 phosphorylation of RGC-32 at Threonine 91. These findings identify RGC-32 as a substrate and regulator of CDC2.
The present invention exploits the surprising discovery that RGC-32 mRNA and protein are expressed in colon carcinoma and other types of cancer, although it does not appear to be expressed at the protein level in normal, surrounding tissue. Indeed, both mRNA levels and protein levels of RGC-32 are increased in tumor tissues. Specifically, immunohistochemistry analysis revealed that RGC-32 is localized in the malignant epithelial cells, but not in adjacent non-neoplastic colonic epithelium, in colon carcinoma. RGC-32 protein was overexpressed in tumor cells that also showed the presence of proliferation marker Ki-67. As a result, increased expression of RGC-32 may be part of the deregulation of the cell cycle that is required for aberrant cell division, neoplastic growth, T-cell proliferation and/or tumor growth and may be useful as a method of detecting various abnormalities.